Psychoneuro 1, psychoneuroimmunologia

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The Journal of Neuroscience, August 5, 2009
•
29(31):9839 –9849 •
9839
Neurobiology of Disease
Systemic Lipopolysaccharide Protects the Brain from
Ischemic Injury by Reprogramming the Response of the
Brain to Stroke: A Critical Role for IRF3
Brenda Marsh,
1
Susan L. Stevens,
1
Amy E. B. Packard,
1
Banu Gopalan,
3
Brian Hunter,
1
Philberta Y. Leung,
1
Christina A. Harrington,
2
and Mary P. Stenzel-Poore
1
1
Department of Molecular Microbiology and Immunology and
2
Vaccine and Gene Therapy Institute, Oregon Health & Science University, Portland,
Oregon 97239, and
3
Genomic Medicine Institute, Cleveland Clinic, Cleveland, Ohio 44195
Lipopolysaccharide (LPS) preconditioning provides neuroprotection against subsequent cerebral ischemic injury through activation of
its receptor, Toll-like receptor 4 (TLR4). Paradoxically, TLR activation by endogenous ligands after ischemia worsens stroke damage.
Here, we define a novel, protective role for TLRs after ischemia in the context of LPS preconditioning. Microarray analysis of brains
collected 24 h after stroke revealed a unique set of upregulated genes in LPS-pretreated animals. Promoter analysis of the unique gene set
identified an overrepresentation of type I interferon (IFN)-associated transcriptional regulatory elements. This finding suggested the
presence of type I IFNs or interferon regulatory factors (IRFs), which upregulate interferon-stimulated genes. Upregulation of IFN
was
confirmed by real-time reverse transcription-PCR. Direct administration of IFN
intracerebroventricularly at the time of stroke was
sufficient for neuroprotection. TLR4 can induce both IFN
and interferon-stimulated genes through its adapter molecule Toll/interleu-
(TRIF) and the IRF3 transcription factor. We show in oxygen glucose depriva-
tion of cortical neurons, an
in vitro
model of stroke, that activation of TRIF after stroke reduces neuronal death. Furthermore, mice
lacking IRF3 were not protected by LPS preconditioning in our
in vivo
model. Our studies constitute the first demonstration of the
neuroprotective capacity of TRIF/IRF3 signaling and suggest that interferon-stimulated genes, whether induced by IFN
or by enhanced
TLR signaling to IRF3, are a potent means of protecting the brain against ischemic damage.
Introduction
It is increasingly clear that Toll-like receptor (TLR) signaling
worsens stroke injury. Mice lacking TLR2 or TLR4 are less sus-
ceptible to damage in multiple models of cerebral ischemia (Cao
et al., 2007; Lehnardt et al., 2007; Ziegler et al., 2007). TLRs are
expressed by microglia, astrocytes, and endothelial cells and are
activated by the damage-associated molecules HSP70 (TLR4)
and HMGB1 (TLR2 and TLR4), present in the brain after isch-
emia (Kinouchi et al., 1993a,b; Faraco et al., 2007). TLR activa-
tion induces production of the inflammatory molecules tumor
necrosis factor
in several models of cerebral ischemia (Tasaki et al., 1997; Rosen-
zweig et al., 2004; Hickey et al., 2007). LPS-induced tolerance to
ischemic injury mirrors the phenomenon of LPS-induced toler-
ance to LPS. Initial exposure of macrophages to LPS induces
proinflammatory TNF
, but, during subsequent exposure to
production is reduced markedly as a result of dis-
rupted signaling through the TLR4 adaptor molecule MyD88
(West and Heagy, 2002; Fan and Cook, 2004; Liew et al., 2005).
Conversely, macrophages produce little interferon
(IFN
) dur-
, and inducible nitric oxide syn-
thase and other cytotoxic mediators that increase tissue damage.
Although TLR4 activation after stroke exacerbates injury, ac-
tivation of TLR4 before stroke protects the brain from damage.
Systemic administration of lipopolysaccharide (LPS), a potent
TLR4 ligand of bacterial origin, renders animals tolerant to injury
), IL1
production during
secondary exposure (Broad et al., 2007), suggesting upregulated
TLR4 signaling through the Toll/interleukin receptor domain-
containing adaptor-inducing IFN
(TRIF) adaptor molecule.
Thus, pretreatment with LPSmay cause cells to switch their dom-
inant TLR4 signaling pathway.
TLR4 signaling through TRIF induces IFN
via activation of
, administered sys-
temically, reduces ischemic brain damage (Liu et al., 2002;
Veldhuis et al., 2003), likely through activation of interferon-
stimulated genes (ISGs). IRF3 itself may have similar neuro-
protective effects. IRF3 binds to interferon-stimulated response
elements (ISREs) within gene promoters, increasing the expres-
sion of many ISGs to the same extent of that elicited by type I IFNs
(Nakaya et al., 2001). Hence, activation of IRF3 may indepen-
dently result in protection from ischemic stroke. Thus, enhanced
Received May 26, 2009; revised June 29, 2009; accepted June 30, 2009.
This work was supported by National Institutes of Health Grant R01 NS050567 (M.P.S.-P.). We thank Jo-Lynn
Boule, Eric Tobar, Delfina Homen, Tao Yang, and Dr. Nikola Lessov for excellent technical support and Dr. Roger
Simon for constructive discussion. Microarray assays were performed in the Affymetrix Microarray Core of the
Oregon Health & Science University Gene Microarray Shared Resource.
Correspondence should be addressed to Dr. Mary P. Stenzel-Poore, Department of Molecular Microbiology and
Immunology, L220, Oregon Health & Science University, 3181 Sam Jackson Park Road, Portland, OR 97239. E-mail:
poorem@ohsu.edu.
DOI:10.1523/JNEUROSCI.2496-09.2009
Copyright © 2009 Society for Neuroscience 0270-6474/09/299839-11$15.00/0
kin receptor domain-containing adaptor-inducing IFN
LPS, TNF
ing initial exposure to LPS but enhance IFN
(TNF
the interferon regulatory factor IRF3. IFN
9840
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J. Neurosci., August 5, 2009
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29(31):9839 –9849
Marsh et al.
•
IRF3 Is Required for LPS Preconditioning
would be expected to contribute to
neuroprotection.
We propose that pretreatment or pre-
conditioning with LPS changes the cel-
lular environment such that subsequent
activation of TLR4 increases signaling via
TRIF to IRF3 and upregulates the neuro-
protective cytokine IFN
. Thus, in this way,
LPS preconditioningmay reprogram subse-
quent activation of TLR4 during ischemia,
which leads to an increase in neuroprotec-
tive type I IFN signaling. Here we provide
evidence for such reprogramming and its
neuroprotective consequences.
25 g)
were purchased from The Jackson Laboratory.
IFN
knock-out mice were kindly provided by
Dr. Leanderson (Lund University, Lund, Swe-
den). IRF3 knock-out mice were procured
from RIKEN BioResource Center (Tsukuba,
Japan). Both strains were backcrossed onto the
C57BL/6 background for at least eight genera-
tions. All mice were housed in an American
Association for Laboratory Animal Care-
approved facility. Procedures were conducted
according to Oregon Health and Science Uni-
versity, Institutional Animal Care and Use
Committee, and National Institutes of Health
guidelines.
LPS treatment.
Mice were given a 200
l in-
traperitoneal injection of saline or LPS [0.2–1.0
mg/kg;
Escherichia coli
serotype
0111:B4
; cata-
log #L2630, purified by phenol extraction, pro-
tein content
3% (Sigma)]. Each new lot of
LPS was titrated to determine the optimal dose
that confers neuroprotection in the particular
strain of mouse being tested.
Middle cerebral artery occlusion.
Mice were
anesthetized with 4% halothane and subjected
to middle cerebral artery occlusion (MCAO)
using the monofilament suture method described previously (Stevens et
al., 2002). Briefly, a silicone-coated 8-0 monofilament nylon surgical
suture was threaded through the external carotid artery to the internal
carotid artery to block themiddle cerebral artery andmaintained intralu-
minally for 40, 45, or 60 min. Duration of occlusion was based on pilot
studies performed to determine the time necessary to obtain an in-
farct size that is between 35 and 45% in the control groups of mice. It
is well known that genetic background can influence ischemic out-
come and thereby affect infarct size. The suture was then removed to
restore blood flow. Cerebral blood flow (CBF) was monitored
throughout surgery by laser Doppler flowmetry. The mean CBF dur-
ing occlusion was between 10 and 17% of baseline in each of the
studies presented. Mice that did not maintain a CBF drop within the
norm of the group during the occlusion were excluded (
4 per
time point) were killed, and cortical brain tissue was collected. RNA was isolated and hybridized to Affymetrix gene chips
(MOE430).
A
, Graph represents the number of genes differentially regulated in LPS- or saline-treated mice compared with un-
handled controls. Time of subsequent stroke is denoted with a black arrow.
B
, Putative biological functions were assigned to the
regulated genes using available public databases and published literature.
6) was included as
unhandled controls. Under RNase-free conditions, a 1 mm section was
removed (4 mm from rostral end) to determine the area of infarct based
on TTC staining. The ipsilateral cortex region from the frontal 4 mmwas
isolated and snap frozen in liquid nitrogen.
RNA isolation.
Total RNA was isolated using the Qiagen RNeasy Lipid
Mini kit. RNA from individual animals was hybridized to single arrays as
described below.
GeneChip expression analyses.
Microarray assays were performed in the
Affymetrix Microarray Core of the Oregon Health and Science Univer-
sity Gene Microarray Shared Resource. RNA samples were labeled using
the NuGEN Ovation Biotin RNA Amplification and Labeling System_V1.
Hybridization was performed as described in the Affymetrix technical
4% of all
animals in the combined studies). Body temperature was maintained
at 37°C with a thermostat-controlled heating pad. The survival rate
for the MCAO procedure was
80%.
Infarct evaluation.
To visualize the region of infarction, 6
1mm
coronal midsections were placed in 1.5% 2,3,5 triphenyltetrazolium
chloride (TTC) in 0.9% PBS and stained at 37°C for 15 min. The infarct
size was determined from computer-scanned images of the hemi-
spheres using NIH Image analyses. To account for edema within the
infarct region, infarct area for each sectionwas computed indirectly as
follows: 100
(contralateral hemisphere area
area of live tissue on
TLR4 signaling to TRIF–IRF3–IFN
Materials and Methods
Mice.
C57BL/6 mice (male, 8–12 weeks,
Figure 1.
Systemic administration of LPS induces early gene regulation in the brain, consistent with an inflammatory response.
C57BL/6micewere administered saline or LPS (0.2mg/kg, i.p.). At varying times (3, 24, and 72 h) after treatment, mice (
n
ipsilateral hemisphere)/(contralateral hemisphere area) (Swanson et
al., 1990).
Experimental design for gene expression studies.
C57BL/6 mice were
divided into 10 groups with four animals per group: groups 1–3 received
a saline injection and were killed at 3, 24, and 72 h, respectively. Groups
4–6 received an LPS injection and were killed at 3, 24, and 72 h, respec-
tively. Groups 7 and 8 received a saline injection, followed 72 h later with
a 45minMCAO. Group 9 and 10 received an LPS injection, followed 72 h
later with a 45 minMCAO. Groups 7 and 9 were killed at 3 h after start of
occlusion with groups 8 and 10 killed 24 h after start of occlusion. At the
time of the mice were killed, the animals were anesthetized and then
perfused with heparinized saline. One group (
n
Marsh et al.
•
IRF3 Is Required for LPS Preconditioning
J. Neurosci., August 5, 2009
•
29(31):9839 –9849
• 9841
manual with modifications as recommended for the Ovation labeling
protocol. Labeled cRNA target was quality checked based on yield and
size distribution. Quality-tested samples were hybridized to theMOE430
2.0 array. The array image was processed with Affymetrix GeneChip
Operating Software. Arrays which did not meet empirically defined cut-
offs within the core facility were remade and hybridized to fresh arrays.
Data were normalized using the robust multichip average method
(Irizarry et al., 2003). The normalized data were then analyzed using a
two-way ANOVA model for each gene, using conditions and time as
groups.
Post hoc
comparisons were made using the unhandled mice as a
control group.
p
values were adjusted for multiple comparisons using the
Hochberg and Benjamini method (Hochberg and Benjamini, 1990).
Genes were considered significantly regulated if the adjusted
p
value was
Table 1. Genes differentially regulated 72 h after LPS preconditioning (time of
stroke)
Title
Symbol
Fold change
Serum amyloid A3
Saa3
6.48
Topoisomerase (DNA) II
Top2a
2.14
UDP glucuronosyltransferase 2 family, polypeptide B37
Ugt2b37
3.53
RIKEN cDNA 4930440C22 gene
2.24
RIKEN cDNA 4930554P06 gene
2.35
0.05, and the fold change in regulation was greater than or equal to 2.
Transcriptional regulatory network analysis.
Using the Web-based pro-
gram Promoter Analysis and Interaction Network Toolset (PAINT) ver-
sion 3.5 (Vadigepalli et al., 2003), we examined the predicted regulatory
elements associated with the unique gene regulation identified by mi-
croarray. In brief, using PAINT, we obtained the 5000 bp upstream se-
quence for the transcripts represented on the MOE430 Affymetrix gene
chip (33,635 transcripts were identified with 5000 bp of upstream se-
quence). PAINT identified putative transcription factor binding se-
quences [transcriptional regulatory elements (TREs)] in these upstream
sequences using the TRANSFAC PROdatabase version 10.4. This pool of
genes and identified TREs was used as our reference comparison group.
The statistical component of PAINT (false discovery rate adjusted
p
value
set at
0.2) was used to determine the overrepresented TREs in individ-
ual gene clusters compared with the reference comparison group (i.e.,
uniquely expressed genes in LPS-preconditioned mice compared with
33,635 member reference group).
Intracerebral ventricular injection of IFN
during MCAO.
Recombi-
(Cell Sciences) or vehicle [artificial CSF (aCSF)]
was injected into the left lateral ventricle as described previously (Meller
et al., 2005). Injections (1
(200 U) or aCSF were
administered immediately before and after surgery (60 min MCAO).
Infarct volume was measured 24 h after stroke.
Quantitative real-time PCR for IFN
l) of either rmIFN
.
RNAwas treatedwithDNase and
transcribed into cDNA using the Omniscript RT kit (Qiagen). Real-time
reverse transcription-PCRs were performed using TaqMan PCR Master
Mix (Applied Biosystems). For IFN
Figure 2.
LPS preconditioning induces a unique set of genes in response to MCAO. C57BL/6
mice were preconditioned with LPS (0.2 mg/kg) or saline 72 h before MCAO (45 min). At 3 or
24 h after MCAO, mice (
n
4 per time point) were killed, and the ipsilateral cortical brain tissue
was collected. RNA was isolated and hybridized to Affymetrix gene chips (MOE430). Venn dia-
gram showing the number of genes differentially regulated in each condition compared with
unhandled controls. Arrows indicate the direction of regulation.
, TaqMan Gene Expression Assay
was used (Mm00439546_S1; Applied Biosystems).
Primers and probe for
-actin were obtained from Integrated DNA
Technologies: forward, 5
-AGAGGGAAATCGTGCGTGAC-3
; reverse,
; probe, CACTGCCGCATC-
CTCTTCCTCCC. Samples were run on an ABI-Prism 7700 (Applied
Biosystems). Results were analyzed using Applied Biosystems sequence
detection software. The relative quantitation of IFN
-CAATAGTGATGACCTGGCCGT-3
normoxic incubator. Control plates were kept in the normoxic incubator
during the OGD interval.
Cell death evaluation
in vitro. Cell death
in vitro
was examined 24 h
after OGD by means of fluorescent, cell-permeable, DNA-binding dyes:
propidium iodide (PI), as an indicator of cell death, and 4
was determined
using the comparative cycle threshold (CT) method (2
CT
) described
in Applied Biosystems User Bulletin #2. Results were normalized to
,6-diamidino-
2-phenylindole (DAPI), as an indicator of the total number of cells.
Coverslips were incubated with PI (1.5
-actin and presented relative to unhandled mice. All reactions were
performed in triplicate.
Oxygen glucose deprivation
in vitro. Primary mouse mixed cortical
cultures were prepared from embryonic day 15 to 17 mouse fetuses.
Cortices were dissected and dissociated with trypsin-EDTA (Invitrogen)
and plated at a density of 4.5
g/ml; Sigma) for 5 min, washed
with PBS, and fixed for 30 min in 10% Formalin. Coverslips were
mounted on slides with Fluoromount-G mounting medium containing
DAPI (Southern Biotechnology Associates). Stained cells were visualized
with a fluorescent microscope (Leica) and analyzed using Metmorph7
software (Molecular Devices). The number of PI- and DAPI-stained cells
were counted in two random fields of view on each coverslip, and per-
centage death was calculated as mean (PI)/(DAPI)
10
5
cells/ml onto coverslips coated with
poly-
L
-ornithine (15 mg/L). Cells were cultured in Neurobasal media
(containing 4.5 g/L glucose; supplemented with Glutamax and B27-AO;
Invitrogen) for 5 d before each experiment. Cultures consisted of
60%
neurons (range, 53–66%) as determined by staining for neuronal-
specific nuclear protein (Millipore Bioscience Research Reagents), with
100 per field of
view. Each treatment was performed with triplicate coverslips within an
experiment, and the entire experiment was repeated three or more times.
Results
Systemic administration of LPS induces an inflammatory
response in the brain
As we have shown previously, systemic administration of LPS
(0.2mg/kg) given 3 d beforeMCAO substantially attenuates isch-
emic damage (Rosenzweig et al., 2004, 2007). To begin to eluci-
date possible mechanisms of neuroprotection, we isolated RNA
from the cortex of LPS-treated and control mice at time points
5% microglia (tomato lectin
;
Vector Laboratories). Oxygen glucose deprivation (OGD) was performed by
removal of the culture medium and replacement with Dulbecco’s PBS (In-
vitrogen), followed by incubation in an anaerobic atmosphere of 85% N
2
,
10% CO
2
, and 5% H
2
at 37°C for 3 h. The anaerobic conditions within
the chamber were monitored using an electronic oxygen/hydrogen ana-
lyzer (Coy Laboratories). OGD was terminated by replacement of the
exposure mediumwith Neurobasal medium (containing 4.5 g/L glucose;
supplemented with Glutamax and B27-AO) and return of the cells to a
5% astrocytes (GFAP
; Sigma) and
nant mouse (rm) IFN
Mix for mouse IFN
5
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29(31):9839 –9849
Marsh et al.
•
IRF3 Is Required for LPS Preconditioning
Table 2. Genes regulated 24 h after stroke only in LPS-preconditioned mice
Symbol
LPS at 3 h
a,b
LPS at 24 h LPS at 72 h LPSMCAO at 3 h LPSMCAO at 24 h
Apoptosis/cell cycle
CD274 antigen
Cd274
11.60
NS
NS
NS
2.48
GLI pathogenesis-related 1 (glioma)
Glipr1
NS
NS
NS
NS
2.34
Caspase 8
Casp8
NS
NS
NS
NS
2.01
Cell movement/cell adhesion
Filamin binding LIM protein 1
Fblim1
NS
NS
NS
NS
2.44
Selectin, endothelial cell
Sele
1.98
NS
NS
1.66
2.37
Neurogenic differentiation 4
Neurod4
NS
1.60
NS
NS
2.33
Kelch-like 6 (Drosophila)
Klhl6
NS
NS
NS
NS
2.18
Glycoprotein (transmembrane) nmb
Gpnmb
NS
NS
NS
NS
2.11
Claudin 1
Cldn1
NS
NS
NS
NS
2.03
M-phase phosphoprotein 1
Mphosph1
NS
NS
NS
NS
2.02
PDZ and LIM domain 5
Pdlim5
NS
NS
NS
1.73
2.01
Protocadherin 20
Pcdh20
NS
NS
NS
NS
2.20
Protocadherin 21
Pcdh21
NS
NS
NS
NS
2.37
Coagulation
Coagulation factor V
F5
NS
NS
NS
NS
3.12
Coagulation factor XIII, A1 subunit
F13a1
NS
NS
NS
NS
2.60
Hepatocyte growth factor
Hgf
NS
NS
NS
NS
2.60
Protein S (
)
Pros1
NS
NS
NS
NS
2.41
Defense response
Radical
S
-adenosyl methionine domain containing 2
Rsad2, VIPERIN 33.06
3.01
NS
NS
3.84
Killer cell lectin-like receptor subfamily B member 1F
Klrb1f
NS
4.77
NS
NS
3.77
Interferon inducible GTPase 1
Iigp1
23.33
NS
NS
NS
3.56
CD52 antigen
Cd52
NS
2.37
NS
NS
3.33
Fc receptor, IgG, high affinity I
Fcgr1
NS
2.77
NS
NS
3.09
Interferon-induced protein with tetratricopeptide repeats 1
Ifit1
19.84
4.03
NS
NS
3.06
Guanylate nucleotide binding protein 3
Gbp3
7.09
2.35
NS
NS
2.96
SLAM family member 9
Slamf9
NS
1.53
NS
NS
2.92
Protein tyrosine phosphatase, receptor type, C
Ptprc, B220
NS
1.94
NS
1.58
2.65
Transporter 1, ATP-binding cassette, subfamily B (MDR/TAP)
Tap1
3.42
1.78
NS
NS
2.65
Histocompatibility 2, Q region locus 1
H2-Q1
7.45
2.84
NS
2.48
2.60
PYD and CARD domain containing
Pycard
NS
2.11
NS
NS
2.58
Lymphocyte cytosolic protein 2
Lcp2
1.79
NS
NS
NS
2.51
Fc receptor, IgE, high affinity I,
polypeptide
Fcer1g
NS
1.87
NS
NS
2.49
2-5 oligoadenylate synthetase-like 2
Oasl2
5.05
3.87
NS
NS
2.48
Phospholipid scramblase 2
Plscr2
3.46
NS
NS
NS
2.47
Interferon -induced GTPase
Igtp
4.35
1.78
NS
NS
2.46
Neutrophilic granule protein
Ngp
NS
NS
NS
NS
2.44
Interferon-induced transmembrane protein 6
Ifitm6
NS
NS
NS
NS
2.44
Complement component 1, q subcomponent,
polypeptide
C1qb
NS
1.54
NS
NS
2.42
Toll-like receptor 4
Tlr4
NS
NS
NS
NS
2.42
Myxovirus (influenza virus) resistance 1
Mx1
11.10
2.43
NS
NS
2.34
Lymphocyte cytosolic protein 1
Lcp1
NS
1.60
NS
NS
2.29
DEAD (Asp-Glu-Ala-Asp) box polypeptide 58
Ddx58, RIG1
3.59
1.79
NS
NS
2.23
Interleukin 1
Il1b
4.69
NS
NS
2.08
2.20
Leukocyte Ig-like receptor, subfamily B member 3
Lilrb3
NS
1.54
NS
NS
2.13
Histocompatibility 2, D region
H2-L
1.55
2.03
1.63
1.61
2.13
Stabilin 1
Stab1
NS
NS
NS
NS
2.12
Histocompatibility 2, K1, K region
H2-K1
2.01
2.38
1.74
1.86
2.11
Protein tyrosine phosphatase, non-receptor type 6
Ptpn6,Shp1 NS
NS
NS
NS
2.11
E74-like factor 1
Elf1
NS
1.98
NS
1.63
2.09
Interferon-induced protein 35
Ifi35
3.27
2.06
NS
NS
2.08
SAM domain, SH3 domain and nuclear localization signals, 1
Samsn1
1.91
NS
NS
1.54
2.08
Histocompatibility 2, D region locus 1
H2-D1
NS
2.05
1.62
1.60
2.08
H-2 class I histocompatibility antigen, Q7 chain precursor
QA-2
5.26
3.89
NS
3.08
2.08
C-type lectin domain family 14, member a
Clec14a
2.36
NS
NS
NS
2.07
C-type lectin domain family 5, member a
Clec5a
NS
NS
NS
NS
2.06
Interferon-induced protein with tetratricopeptide repeats 3
Ifit3
5.41
2.42
NS
NS
2.06
Proteosome subunit, type 8
Psmb8
2.56
2.03
NS
NS
2.05
Neutrophil cytosolic factor 1
Ncf1
1.51
NS
NS
NS
2.03
Lymphocyte antigen 6 complex, locus A
Ly6a
1.79
1.88
NS
NS
2.01
Signal peptide, CUB domain, EGF-like 1
Scube1
NS
NS
NS
NS
2.03
Corticotropin releasing hormone
Crh
NS
NS
NS
NS
2.10
(
Table continues
.)
Marsh et al.
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IRF3 Is Required for LPS Preconditioning
J. Neurosci., August 5, 2009
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29(31):9839 –9849
• 9843
Table 2. Continued
Symbol
LPS at 3 h
a,b
LPS at 24 h LPS at 72 h LPSMCAO at 3 h LPSMCAO at 24 h
Metabolic processes
Klotho
Kl
NS
NS
NS
NS
2.48
Hexokinase 2
Hk2
NS
NS
NS
NS
2.26
Ethanolamine kinase 1
Etnk1
NS
NS
NS
2.89
2.24
Carbonic anhydrase 13
Car13
NS
NS
NS
NS
2.16
Centromere protein A
Cenpa
NS
NS
NS
NS
2.12
Phosphodiesterase 3A, cGMP inhibited
Pde3a
NS
2.19
NS
NS
2.08
Folate receptor 1 (adult)
Folr1
NS
NS
NS
NS
2.04
AMP deaminase 3
Ampd3
NS
NS
NS
NS
2.02
Miscellaneous cell processes
Schlafen 2
Slfn2
4.00
2.18
NS
NS
3.35
Calmodulin-like 4
Calml4
NS
NS
NS
NS
2.50
Ecotropic viral integration site 2b
Evi2b
NS
NS
NS
NS
2.49
Estrogen receptor 1 ( )
Esr1
NS
NS
NS
2.35
2.09
Luc7 homolog (
Saccharomyces / cerevisiae
)-like
Luc7l
NS
NS
NS
NS
2.04
Protein/RNA processing
Ubiquitin specific peptidase 18
Usp18
12.83
3.83
NS
NS
2.89
RIKEN cDNA 5430435G22 gene
NS
NS
NS
NS
2.75
Ribosomal protein L7
Rpl7
NS
2.11
NS
NS
2.75
Heat shock protein 8
Hspb8
NS
NS
NS
1.70
2.42
Serine (or cysteine) peptidase inhibitor, clade H, member 1
Serpinh1
NS
NS
NS
1.70
2.33
-galactosyl-1,3)-
N
-acetyl-
galactosaminide -2,6-sialyltransferase 2
-
N
-acetyl-neuraminyl-2,3-
St6galnac2
NS
NS
NS
NS
2.31
UDP-GlcNAc: Gal -1,3-N-acetylglucosaminyltransferase 5
B3gnt5
NS
NS
NS
NS
2.27
Phospholipase A2, group IVA
Pla2g4a
NS
NS
NS
NS
2.27
Z-DNA binding protein 1
Zbp1
3.50
3.40
NS
NS
2.27
A disintegrin-like and metallopeptidase with thrombospondin type 1 motif, 5 Adamts5
NS
NS
NS
NS
2.13
Translocating chain-associating membrane protein 2
Tram2
NS
NS
NS
NS
2.08
IMP4, U3 small nucleolar ribonucleoprotein
Imp4
NS
NS
NS
NS
2.08
Ribosomal protein S25
Rps25
NS
NS
NS
NS
2.08
Ubiquitin-like, containing PHD and RING finger domains, 1
Uhrf1
NS
NS
NS
NS
2.06
cDNA sequence BC099439
BC099439
NS
NS
NS
NS
2.05
DnaJ (Hsp40) homolog, subfamily B, member 5
Dnajb5
NS
NS
NS
NS
2.04
Signal transduction
Receptor transporter protein 4
Rtp4
3.83
3.46
NS
NS
2.92
Component of Sp100-rs
Csprs
NS
2.95
NS
NS
2.36
Rho GTPase activating protein 30
Arhgap30
NS
NS
NS
NS
2.33
Adenylate cyclase 7
Adcy7
NS
NS
NS
NS
2.28
Guanine nucleotide binding protein, 14
Gna14
NS
NS
NS
NS
2.28
Vomeronasal 1 receptor, A1
V1ra1
NS
2.52
NS
NS
2.22
Fibrinogen-like protein 2
Fgl2
NS
NS
NS
NS
2.14
Ras homolog gene family, member C
Rhoc
1.66
NS
NS
1.55
2.12
Pleckstrin homology, Sec7 and coiled/coil domains 4
Pscd4
NS
1.52
NS
NS
2.07
Pleckstrin homology domain containing, family G member 2
Plekhg2
NS
NS
NS
NS
2.05
Transcription
Reduced expression 2
Rex2
NS
NS
NS
NS
2.28
MyoD family inhibitor domain containing
Mdfic
NS
NS
NS
NS
2.24
Leucine rich repeat (in FLII) interacting protein 1
Lrrfip1
NS
NS
NS
2.05
2.16
Bromodomain adjacent to zinc finger domain 1A
Baz1a
NS
NS
NS
2.28
2.05
Ladybird homeobox 1 homolog (
Drosophila
) corepressor 1
Lbxcor1
NS
NS
NS
1.87
2.04
Annexin A11
Anxa11
NS
NS
NS
NS
2.02
Tripartite motif protein 30
Trim30
8.15
2.13
NS
NS
2.02
Inversin
Invs
NS
2.11
NS
NS
2.02
Transport
Transthyretin
Ttr
NS
NS
NS
NS
12.74
Translocator protein
Tspo
NS
2.16
NS
NS
2.76
Stanniocalcin 2
Stc2
NS
NS
NS
1.97
2.64
Transient receptor potential cation channel, subfamily M, member 3
Trpm3
NS
NS
NS
NS
2.63
Potassium voltage-gated channel, Isk-related subfamily, gene 2
Kcne2
NS
NS
NS
NS
2.41
Transferrin
Trf
NS
NS
NS
NS
2.37
Chloride channel calcium activated 1///chloride channel calcium activated 2 Clca1///Clca2 NS
NS
NS
NS
2.26
Mannose receptor, C type 1
Mrc1
NS
NS
NS
NS
2.25
Cysteine-rich hydrophobic domain 2
Chic2
NS
NS
NS
NS
2.10
Exocyst complex component 6
Exoc6
NS
NS
NS
NS
2.10
SEH1-like (
S. cerevisiae
)
Seh1l
NS
NS
NS
NS
2.08
(
Table continues
.)
ST6 (
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